Dynamics of H3K36me3 in demethylase deletion strains.

<p>The relative amount of histone H3 (<b>A, C, E, G, I</b>) and H3K36me3 (<b>B, D, F, H, J</b>) was determined at 2.6 kb of the <i>GAL-VPS13</i> upon transcription repression in wild type (<b>A</b> and <b>B</b>), <i>rph1Δ</i> (<b>C</b> and <b>D</b>), <i>jhd1Δ</i> (<b>E</b> and <b>F</b>), <i>gis1Δ</i> (<b>G</b> and <b>H</b>) and <i>rph1Δjhd1Δgis1Δ</i> triple deletion mutant (ΔΔΔ <b>I</b> and <b>J</b>) strains. All samples were quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120200#pone.0120200.g001" target="_blank">Fig. 1</a>.</p

Distribution and Maintenance of Histone H3 Lysine 36 Trimethylation in Transcribed Locus

<div><p>Post-translational modifications of core histones play an important role in the epigenetic regulation of chromatin dynamics and gene expression. In <i>Saccharomyces cerevisiae</i> methylation marks at K4, K36, and K79 of histone H3 are associated with gene transcription. Although Set2-mediated H3K36 methylation is enriched throughout the coding region of active genes and prevents aberrant transcriptional initiation within coding sequences, it is not known if transcription of one locus impacts the methylation pattern of neighbouring areas and for how long H3K36 methylation is maintained after transcription termination. Our results demonstrate that H3K36 methylation is restricted to the transcribed sequence only and the modification does not spread to adjacent loci downstream from transcription termination site. We also show that H3K36 trimethylation mark persists in the locus for at least 60 minutes after transcription inhibition, suggesting a short epigenetic memory for recently occurred transcriptional activity. Our results indicate that both replication-dependent exchange of nucleosomes and the activity of histone demethylases Rph1, Jhd1 and Gis1 contribute to the turnover of H3K36 methylation upon shut-down of transcription.</p></div

Replication-dependent loss of H3K36me3 after transcriptional repression in wt and <i>rph1Δjhd1Δgis1Δ</i> strains.

<p>The relative amount of histone H3 (<b>A</b> and <b>C</b>) and H3K36me3 (<b>B</b> and <b>D</b>) was determined upon glucose-mediated transcriptional repression at 2.6 kb in the coding region of <i>GAL-VPS13</i> in G1-arrested wt (<b>A</b> and <b>B</b>), and in <i>rph1Δjhd1Δgis1Δ</i> cells (ΔΔΔ; <b>C</b> and <b>D</b>). (<b>E</b>) Comparison of H3K36me3 turnover in wt and <i>rph1Δjhd1Δgis1Δ</i> strains grown asynchronously, or kept G1-arrested throughout the experiment. The Student <i>t</i> test was used to evaluate the statistical significance of differences between indicated samples. * indicates p<0.05; ** indicates p<0.005; *** indicates p<0.0001. All samples were quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120200#pone.0120200.g001" target="_blank">Fig. 1</a>. Cell cycle profiles of G1-arrested wt and <i>rph1Δjhd1Δgis1Δ</i> strains are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120200#pone.0120200.s002" target="_blank">S2 Fig.</a></p

Yeast strains.

<p>Yeast strains.</p

Distribution of H3K36me3 in <i>GAL-VPS13</i> locus.

<p>(<b>A</b>) Schematic representation of the 9435 bp long <i>GAL-VPS13</i> locus. The <i>GAL-VPS13-3kb–term</i> strain contains the <i>FBA1</i> transcription termination region inserted at 3 kb from the <i>VPS13</i> promoter (3 kb-terminator, black rectangle). Vertical lines beneath the gene indicate the positions of PCR probes 2.6 kb (a), 3 kb (b), 3.6 kb (c) and 4 kb (d) downstream from the start-codon of <i>GAL-VPS13</i>. (<b>B</b>) ChIP assay followed by qPCR was used to determine the relative amount of histone H3 in the coding region of <i>GAL-VPS13</i> upon transcriptional activation in galactose (black bars, Gal) and repression in glucose (grey bars, Glu). (<b>C</b>) The relative amount of H3K36me3 was determined in the coding region of <i>GAL-VPS13</i> upon transcriptional activation in galactose (black bars, Gal) and repression in glucose (grey bars, Glu). (<b>D</b>) The occupancy of Set2 upon transcriptional activation in galactose (black bars, Gal) and inactivation in glucose (grey bars, Glu) in the coding region of <i>GAL-VPS13</i>. In all assays, the ChIP signal obtained from nontranscribed region of the right arm telomere of chromosome VI (Tel6) was set as 1 and all samples are presented as relative to that. In addition, the H3K36 trimethylation signal in C was normalised to total H3 occupancy. Error bars show standard error of at least 3 independent experiments.</p